The Florence outfit fought hard but lost 1-0 to Juve in Serie A on Sunday to fall 10 points behind Napoli in the race for Champions League football next season.
"I want my team to pick up where they left off in the second half against Juventus on Sunday," Fiorentina coach Vincenzo Montella said.
"I was satisfied with my team's performance and we have to have confidence in view of Thursday's match.
"Juventus are strong but I think we can have a say."
The Viola have been a force in Italy this season and can approach the tie with confidence as they are the only side to have beaten Juve in Serie A, a 4-2 triumph in October, while they have reached the Coppa Italia final.
In the Europa League, Montella's team topped Group E before dumping out Esbjerg in the last 32.
"I expect a battling Fiorentina," Juventus coach Antonio Conte said. "They will be determined to progress in the competition because they have little to play for in Serie A while we, in contrast, have a third straight Scudetto to work for.
"Fiorentina will try to put all that they have into this competition.
"Montella is a great coach and I think this tie will be a beautiful advert for Italian and European football."
Juve have the extra incentive of getting to the final because it will be held in their own stadium.
Moreover, they are keen to end their drought of European titles with their last coming in 1999, the UEFA Intertoto Cup.
"It would be fantastic to go all the way in this competition, it would allow us to grow from an international standpoint," Conte said.
Sunday's win, courtesy of Kwadwo Asamoah's goal, coupled with Roma's defeat at Napoli allowed Juve to take a giant step towards their third straight league title.
Juve are 14 points clear of Roma at the top of the standings.
"We are doing well but the Scudetto race is not over and there are still 11 games left to play," Conte said."We needed three points on Sunday and we've got that.
"Now we face the Europa League and this becomes the game of our lives."
Fiorentina, who host Juve in the return leg on March 20, will be bolstered by the return of Borja Valero after the midfielder missed Sunday's game through suspension.
The Viola will be without Spanish winger Joaquin, who is sidelined with a muscular injury sustained in training.